The globin family of proteins has been extensively studied and includes hemoglobin which, as potentially useful oxygen carrying therapeutic, has been the focus of much research and development effort. The primary aim of this research is the design and synthesis of a single-chain human hemoglobin which binds oxygen reversibly and cooperatively. A single-chain hemoglobin could be more easily engineered for optimum performance as a blood substitute than the multisubunit complex. The development of a single-chain hemoglobin molecule would be a significant achievement in protein engineering as well as a significant step forward in the development of oxygen delivery therapeutics. A single-chain hemoglobin would also allow for high resolution mutagenesis studies of allostery in a single protein framework, which would expand our understanding of cooperativity in protein function. The proposed research will increase our understanding of the effects of altered protein topology on protein structure, and function in a stringent system (i.e., an allosteric protein). We aim to demonstrate general approaches for altering the order of structural elements in complex proteins, and thereby expand the scope of topological mutation as a protein engineering tool. The specific aims of the proposed research are: (1) to generate a circularly permuted beta-globin modeled on the permuted myoglobin characterized in our previous work; (2) to insert the permuted beta-globin into the G-H loop of alpha-globin to create single-chain alpha/beta dimers; (3) to fuse two alpha/beta dimers to create the target single-chain hemoglobin; (4) to determine the functional (e.g. ligand binding) properties, and thermodynamic stabilities of these novel constructs.